Replies to post #485892 on NorthWest Biotherapeutics Inc (NWBO)

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How Similar is this process to DCVAX?

Whole tumor lysates preparation
To prepare whole tumor cell lysates (WTL) for prophylactic immunization, tumor cell lines were cultured for 4- 5 days in complete DMEM. When cells became confluent, supernatants were collected, and the cells were washed and dissociated using trypsin- EDTA (GIBCO). Tumor cell lines were then resuspended at 5x106 cells/ml in their collected culture supernatant, then lysed by 3 cycles of freeze-thawing. For immunotherapy experiments, syngeneic WTL were prepared from tumors explants of unimmunized tumor- bearing mice. Briefly, tumors from unimmunized mice bearing a tumor 10-12 mm of size were mechanically disaggregated using gen- tleMACS dissociator (Miltenyi Biotec) and digested using the tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer’s protocol. Tumors were incubated for 45 minutes at 37 degrees in a tube rotator for complete digestion. After digestion, tumor cell suspensions were washed with PBS and passed through 100-mm then 70-mm then 30-mm filters. Single cell suspension was depleted of CD45+ cells using anti-CD45 TILs microbeads (Miltenyi Biotec). Tumor cells were then counted and resuspended at 5x106 cells/ml then lysed by 3-4 cycles of freeze-thawing. All prepared Lysates were centrifuged at 12,000 rpm for 15 minutes, and supernatants were passed through 70-mm and 30-mm filters then stored in aliquots at
20C until use. WTL were used for immunotherapy or for BMDC antigen loading at a concentration equivalent to 2.5x105 tumor cells per mice, or at ratio equivalent to 1:10 (DC:tumor cells) respectively.